Isolated nucleic acid molecules encoding isolated peptides which correspond to contiguous amino acids of an SSX molecule or NY-ESO-1 and uses thereof

ABSTRACT

The invention relates to members of the SSX family of genes, as well as their uses. Also a part of the invention are peptides derived from SSX molecules and the NY-ESO-1 molecule, which form complexes with HLA molecules, leading to lysis of cells presenting these complexes, by cytolytic T cells.

RELATED APPLICATION

This application is a divisional of application Ser. No. 09/344,040,filed Jun. 25, 1999, now U.S. Pat. No. 6,548,064, which is acontinuation in part of application Ser. No. 08/851,138 filed on May 5,1997, now U.S. Pat. No. 6,291,658, and of Ser. No. 09/105,839, filedJun. 26, 1998, now U.S. Pat. No. 6,287,756 each of which is incorporatedby reference.

FIELD OF THE INVENTION

This invention relates to the isolation and cloning of genes which aremembers of the “SSX” family, which is discussed herein, and the usesthereof, including determination of cancer. Also a part of the inventionare peptides derived from these SSX genes, as well as from the NY-ESO-1gene. These peptides stimulate proliferation of cytolytic T cells, andthus are useful as markers for presence of disorders such as cancer, forHLA-A2 cells, and as therapeutic agents for treating cancer.

BACKGROUND AND PRIOR ART

It is fairly well established that many pathological conditions, suchas, infections, cancer, autoimmune disorders, etc., are characterized bythe inappropriate expression of certain molecules. These molecules thusserve as “markers” for a particular pathological or abnormal condition.Apart from their use as diagnostic “targets”, i.e., materials to beidentified to diagnose these abnormal conditions, the molecules serve asreagents which can be used to generate diagnostic and/or therapeuticagents. A by no means limiting example of this is the use of cancermarkers to produce antibodies specific to a particular marker. Yetanother non-limiting example is the use of a peptide which complexeswith an MHC molecule, to generate cytolytic T cells against abnormalcells.

Preparation of such materials, of course, presupposes a source of thereagents used to generate these. Purification from cells is onelaborious, far from sure method of doing so. Another preferred method isthe isolation of nucleic acid molecules which encode a particularmarker, followed by the use of the isolated encoding molecule to expressthe desired molecule.

To date, two strategies have been employed for the detection of suchantigens, in e.g., human tumors. These will be referred to as thegenetic approach and the biochemical approach. The genetic approach isexemplified by, e.g., dePlaen et al., Proc. Natl. Sci. USA 85: 2275(1988), incorporated by reference. In this approach, several hundredpools of plasmids of a cDNA library obtained from a tumor aretransfected into recipient cells, such as COS cells, or intoantigen-negative variants of tumor cell lines. Transfectants arescreened for the expression of tumor antigens via their ability toprovoke reactions by anti-tumor cytolytic T cell clones. The biochemicalapproach, exemplified by, e.g., Mandelboim, et al., Nature 369: 69(1994) incorporated by reference, is based on acidic elution of peptideswhich have bound to MHC-class I molecules of tumor cells, followed byreversed-phase high performance liquid chromography (HPLC). Antigenicpeptides are identified after they bind to empty MHC-class I moleculesof mutant cell lines, defective in antigen processing, and inducespecific reactions with cytotoxic T-lymphocytes. These reactions includeinduction of CTL proliferation, TNF release, and lysis of target cells,measurable in an MTT assay, or a ⁵¹Cr release assay.

These two approaches to the molecular definition of antigens have thefollowing disadvantages: first, they are enormously cumbersome,time-consuming and expensive; second, they depend on the establishmentof cytotoxic T cell lines (CTLs) with predefined specificity; and third,their relevance in vivo for the course of the pathology of disease inquestion has not been proven, as the respective CTLs can be obtained notonly from patients with the respective disease, but also from healthyindividuals, depending on their T cell repertoire.

The problems inherent to the two known approaches for the identificationand molecular definition of antigens is best demonstrated by the factthat both methods have, so far, succeeded in defining only very few newantigens in human tumors. See, e.g., van der Bruggen et al., Science254: 1643-1647 (1991); Brichard et al., J. Exp. Med. 178: 489-495(1993); Coulie, et al., J. Exp. Med. 180: 35-42 (1994); Kawakami, etal., Proc. Natl. Acad. Sci. USA 91: 3515-3519 (1994).

Further, the methodologies described rely on the availability ofestablished, permanent cell lines of the cancer type underconsideration. It is very difficult to establish cell lines from certaincancer types, as is shown by, e.g., Oettgen, et al., Immunol. Allerg.Clin. North. Am. 10: 607-637 (1990). It is also known that someepithelial cell type cancers are poorly susceptible to CTLs in vitro,precluding routine analysis. These problems have stimulated the art todevelop additional methodologies for identifying cancer associatedantigens.

One key methodology is described by Sahin, et al., Proc. Natl. Acad.Sci. USA 92: 11810-11913 (1995), incorporated by reference. Also, seeU.S. patent application Ser. No. 08/580,980, and filed on Jan. 3, 1996,and U.S. Pat. No. 5,698,396. All three of these references areincorporated by reference. To summarize, the method involves theexpression of cDNA libraries in a prokaryotic host. (The libraries aresecured from a tumor sample). The expressed libraries are thenimmunoscreened with absorbed and diluted sera, in order to detect thoseantigens which elicit high titer humoral responses. This methodology isknown as the SEREX method (“Serological identification of antigens byRecombinant Expression Cloning”). The methodology has been employed toconfirm expression of previously identified tumor associated antigens,as well as to detect new ones. See the above referenced patentapplications and Sahin, et al., supra, as well as Crew, et al., EMBO J144: 2333-2340 (1995).

The SEREX methodology has been applied to esophageal cancer samples, andan esophageal cancer associated antigen has now been identified, and itsencoding nucleic acid molecule isolated and cloned, as per U.S. patentapplication Ser. No. 08/725,182, filed Oct. 3, 1996, incorporated byreference herein.

The relationship between some of the tumor associated genes and a triadof genes, known as the SSX genes, is under investigation. See Sahin, etal., supra; Tureci, et al., Cancer Res 56:4766-4772 (1996). One of theseSSX genes, referred to as SSX2, was identified, at first, as one of twogenes involved in a chromosomal translocation event (t(X; 18)(p11.2; q11.2)), which is present in 70% of synovial sarcomas. See Clark, et al.,Nature Genetics 7:502-508 (1994); Crew et al., EMBO J 14:2333-2340(1995). It was later found to be expressed in a number of tumor cells,and is now considered to be a tumor associated antigen referred to asHOM-MEL-40 by Tureci, et al, supra. Its expression to date has beenobserved in cancer cells, and normal testis only. Thus parallels othermembers of the “CT” family of tumor antigens, since they are expressedonly in cancer and testis cells. Crew et al. also isolated and clonedthe SSX1 gene, which has 89% nucleotide sequence homology with SSX2.Sequence information for SSX1 and SSX2 is presented as SEQ ID NOS: 1 and2 respectively. See Crew et al., supra. Additional work directed to theidentification of SSX genes has resulted in the identification of SSX3,as is described by DeLeeuw, et al., Cytogenet. Genet 73:179-183 (1996).The fact that SSX presentation parallels other, CT antigens suggested tothe inventors that other SSX genes might be isolated. The parentapplication, supra discloses this work, as does Gure, et al. Int. J.Cancer 72:965-971(1997), incorporated by reference.

With respect to additional literature on the SSX family, most of itrelates to SSX1. See PCT Application W/96 02641A2 to Cooper, et al,detailing work on the determination of synovial sarcoma viadetermination of SSX1 or SSX2. Also note DeLecuw, et al. Hum. Mol. Genet4(6):1097-1099 (1995), also describing synovial sarcoma and SYT-SSX1 orSSX2 translocation. Also see Kawai, et al, N. Engl. J. Med338(3):153-160 (1998); Noguchi, et al. Int. J. Cancer 72(6):995-1002(1997), Hibshoosh, et al., Semin. Oncol 24(5):515-525 (1997), Shipley,et al., Am. J. Pathol. 148(2):559-567 (1996); Fligman, et al. Am. J.Pathol. 147(6); 1592-1599 (1995). Also see Chand, et al., Genomics30(3):545-552 (1995), Brett, et al., Hum. Mol Genet 6(9): 1559-1564(1997), deBruyn, et al, Oncogene (13/3):643-648. The SSX3 gene isdescribed by deLeeuw, et al, Cytogenet Cell Genet 73(3):179-1983 (1966).

Application of a modification of the SEREX technology described suprahas been used, together with other techniques, to clone two, additionalSSX genes, referred to as SSX4 and SSX5 hereafter as well as analternate splice variant of the SSX4 gene. Specifically, while the SEREXmethodology utilizes autologous serum, the methods set forth infra useallogenic serum.

Motif analysis is a tool which permits one to ascertain what regions ofa longer protein may in fact be of particular interest as binders of MHCor HLA molecules. Essentially, one works with an amino acid motif, whichgenerally includes at least two, and sometimes more, defined amino acidsin a sequence of 8-12 amino acids. This motif is then used to screen alonger sequence to determine which sequences within the longer sequenceconstitute peptides which would bind to an HLA or MHC molecule, andpossibly stimulate proliferation of cytolytic T lymphocytes withspecificity to complexes of the peptide and MHC/HLA molecule. Motifsdiffer for different MHC/HLA molecules. Much work has been done in thisarea, but it is ongoing. As will be seen in the disclosure whichfollows, the inventors have used motif analysis to identify peptideswhich bind to HLA molecules, HLA-A2 molecules in particular.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

A human testicular cDNA expression library was obtained, and screened,with serum from a melanoma patient identified as MZ2. See e.g., parentapplication U.S. patent application Ser. No. 08/479,328 incorporated byreference; also see U.S. patent application Ser. No. 08/725,182 alsoincorporated by reference; Sahin, et al., Proc. Natl. Acad. Sci. USA92:11810-11813 (1995). This serum had been treated using the methodologydescribed in these references. Briefly, serum was diluted 1:10, and thenpreabsorbed with transfected E. coli lysate. Following thispreabsorption step, the absorbed serum was diluted 1:10, for a finaldilution of 1:100. Following the final dilution the samples wereincubated overnight at room temperature, with nitrocellulose membranescontaining phage plaques prepared using the methodology referred tosupra. The nitrocellulose membranes were washed, incubated with alkalinephosphatase conjugated goat anti-human Fc_(γ) secondary antibodies, andthe reaction was observed with the substrates 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium. In a secondary screen, anyphagemids which encoded human immunoglobulin were eliminated.

A total of 3.6×10⁵ pfus were screened, resulting in eight positiveclones. Standard sequencing reactions were carried out, and thesequences were compared to sequence banks of known sequences.

Of the eight clones, two were found to code for known autoimmune diseaseassociated molecules, i.e., Golgin-95 (Fritzler, et al., J. Exp. Med.178:49-62 (1993)), and human upstream binding factor (Chan, et al., J.Exp. Med. 174:1239-1244 (1991)). Three other clones were found to encodefor proteins which are widely expressed in human tissue, i.e., ribosomalreceptor, collagen type VI globular domain, and rapamycin bindingprotein. Of the remaining three sequences, one was found to benon-homologous to any known sequence, but was expressed ubiquitously inhuman tissues (this was found via RT-PCR analysis, but details are notprovided herein). The remaining two were found to be identical to fulllength HOM-MEL-40, described in Ser. No. 08/479,328, while the eighthclone was found to be almost identical to “SSX3,” as described byDeLeeuw, et al., Cytogenet. Cell Genet 73:179-183 (1996), differingtherefrom in only two base pair differences in the coding region. Thesedifferences are probably artifactual in nature; however, the clone alsoincluded a 43 base pair 3′-untranslated region.

EXAMPLE 2

In order to carry out Southern blotting experiments, described infra,the SSX genes were amplified, using RT-PCR.

To do this, two primers were prepared using the published SSX2 sequencei.e., MEL-40A:

MEL-40A: (SEQ ID NO: 3) 5′-CACACAGGAT CCATGAACGG AGA, and MEL-40B: (SEQ.ID NO: 4) 5′-CACACAAAGC TTTGAGGGGA GTTACTCGTC ATCSee Crew, et al., EMBO J 14:2333-2340 (1995). Amplification was thencarried out using 0.25 U Taq polymerase in a 25 μl reaction volume,using an annealing temperature of 60° C. A total of 35 cycles werecarried out.

EXAMPLE 3

The RT-PCR methodology described supra was carried out on testiculartotal RNA, and the amplification product was used in southern blottingexperiments.

Genomic DNA was extracted from non-neoplastic tissue samples, and thensubjected to restriction enzyme digestion, using BamHI, Eco RI, orHindIII in separate experiments and then separated on a 0.7% agarosegel, followed by blotting on to nitrocellulose filters. Theamplification products described supra were labeled with ³²P, usingwell-known methods, and the labeled materials were then used as probesunder high stringency conditions (65° C., aqueous buffer), followed byhigh stringency washes, ending with a final wash at 0.2×SSC, 0.2% SDS,65° C.

The Southern blotting revealed more than 10 bands, in each case (i.e.,each of the BamHI, EcoRI, and HindIII digests), strongly suggesting thatthere is a family of SSX genes which contained more than the threeidentified previously. In view of this observation, an approach wasdesigned which combined both PCR cloning, and restriction map analysis,to identify other SSX genes.

EXAMPLE 4

When the sequences of SSX1, 2 and 3 were compared, it was found thatthey shared highly conserved 5′ and 3′ regions, which explained why theolignucleotides of SEQ ID NOS: 3 and 4 were capable of amplifying allthree sequences under the recited conditions, and suggested that thishomology was shared by the family of SSX genes, whatever its size.Hence, the oligonucleotides of SEQ ID NOS: 3 and 4 would be sufficientto amplify the other members of the SSX gene family.

An analysis of the sequences of SSX1, 2 and 3 revealed that SSX1 and 2contained a BglII site which was not shared by SSX3. Similarly, SSX3contained an EcoRV site not shared by the other genes.

In view of this information, testicular cDNA was amplified, using SEQ IDNOS: 3 and 4, as described supra, and was then subjected to BglIIdigestion. Any BglII resistant sequences were then cloned, sequenced,and compared with the known sequences.

This resulted in the identification of two previously unidentifiedsequences, referred to hereafter as SSX4 and SSX5, presented as SEQ IDNOS: 5 and 6 herein. A search of the GenBank database found two clones,identified by Accession Number N24445 and W00507, both of whichconsisted of a sequence-tag-derived cDNA segment. The clone identifiedby N24445 contained the 3′-untranslated region of SSX4, and part of itscoding sequence, while the one identified as W00507 contained a shorterfragment of the 3′-untranslated region of SSX4, and a longer part of thecoding sequence. Specifically, N24445 consists of base 344 of SSX4 (SEQID NO:5), through the 3-end, plus 319 bases 3′ of the stop codon. TheW00507 sequence consists of a 99 base pair sequence, showing no homologyto SSX genes followed by a region identical to nucleotides 280 throughthe end of SEQ ID NO:5, through 67 bases 3′ of the stop codon of SEQ IDNO:1.

Two forms of SSX4 (SEQ ID NO: 5) were identified. One of these lackednucleotides 331 to 466 but was otherwise identical to SSX4 as presentedin SEQ ID NO: 5. As is described infra, the shorter form is analternatively spliced variant.

In Table 1, which follows, the nucleotide and amino acid sequences ofthe 5 known members of the SSX family are compared. One reads the tablehorizontally for nucleotide homology, and vertically for amino acidhomology.

TABLE 1 Nucleotide and amino acid homology among SSX family membersNuclcotide Sequence Homology (%) SSX1 SSX2 SSX3 SSX4 SSX5 SSX1 89.1 89.689.4 88.7 SSX2 78.2 95.1 91.5 92.9 SSX3 77.7 91.0 91.1 92.7 SSX4 79.379.8 80.9 89.8 SSX5 76.6 83.5 84.0 77.7 Amino Acid Sequence Homology (%)Hence, SSX1 and SSX4 share 89.4% homology on the nucleotide level, and79.3% homology on the amino acid level.

When the truncated form of SSX4 is analyzed, it has an amino acidsequence completely different from others, due to alternate splicing andshifting of a downstream open reading frame. The putative protein is 153amino acids long, and the 42 carboxy terminal amino acids show nohomology to the other SSX proteins.

EXAMPLE 5

The genomic organization of the SSX2 genes was then studied. To do this,a genomic human placental library (in lambda phage) was screened, usingthe same protocol and probes described supra in the discussion of thesouthern blotting work. Any positive primary clones were purified, viatwo additional rounds of cloning.

Multiple positive clones were isolated, one of which was partiallysequenced, and identified as the genomic clone of SSX2. A series ofexperiments carrying out standard subcloning and sequencing workfollowed, so as to define the exon-intron boundaries.

The analysis revealed that the SSX2, gene contains six exons, and spansat least 8 kilobases. All defined boundaries were found to observe theconsensus sequence of exon/intron junctions, i.e. GT/AG.

The alternate splice variant of SSX4, discussed supra, was found to lackthe fifth exon in the coding region. This was ascertained by comparingit to the SSX2 genomic clone, and drawing correlations therefrom.

EXAMPLE 6

The expression of individual SSX genes in normal and tumor tissues wasthen examined. This required the construction of specific primers, basedupon the known sequences, and these follow, as SEQ ID NOS: 7-16:

TABLE 2 Gene-specific PCR primer sequences for individual SSX genes SSX1A (5′): 5′-CTAAAGCATCAGAGAAGAGAAGC [nt.  44-66] SSX 1B (3′):5′-AGATCTCTTATTAATCTTCTCAGAAA [nt. 440-65] SSX 2A (5′):5′-GTGCTCAAATACCAGAGAAGATC [nt.  41-63] SSX 2B (3′):5′-TTTTGGGTCCAGATCTCTCGTG [nt. 102-25] SSX 3A (5′):5′-GGAAGAGTGGGAAAAGATGAAAGT [nt. 454-75] SSX 3B (3′):5′-CCCCTTTTGGGTCCAGATATCA [nt. 458-79] SSX 4A (5′):5′-AAATCGTCTATGTGTATATGAAGCT [nt. 133-58] SSX 4B (3′):5′-GGGTCGCTGATCTCTTCATAAAC [nt. 526-48 SSX 5A (5′):5′-GTTCTCAAATACCACAGAAGATG [nt.  39-63] SSX 5B (3′):5′-CTCTGCTGGCTTCTCGGGCCG [nt. 335-54]The specificity of the clones was confirmed by amplifying the previouslyidentified cDNA for SSX1 through SSX5. Taq polymerase was used, at 60°C. for SSX1 and 4, and 65° C. for SSX2, 3 and 5. Each set of primerpairs was found to be specific, except that the SSX2 primers were foundto amplify minute (less than 1/20 of SSX2) amounts of SSX3 plasmid DNA.

Once the specificity was confirmed, the primers were used to analyzetesticular mRNA, using the RT-PCR protocols set forth supra.

The expected PCR products were found in all 5 cases, and amplificationwith the SSX4 pair did result in two amplification products, which isconsistent with alternative splice variants.

The expression of SSX genes in cultured melanocytes was then studied.RT-PCR was carried out, using the protocols set forth supra. No PCRproduct was found. Reamplification resulted in a small amount of SSX4product, including both alternate forms, indicating that SSX4 expressionin cultured melanocytes is inconsistent and is at very low levels whenit occurs.

This analysis was then extended to a panel of twelve melanoma celllines. These results are set forth in the following table.

TABLE 3 SSX expression in melanoma cell lines detected by RT-PCR* SSX1SSX2 SSX3 SSX4 SSX5 MZ2-Mel 2.2 + + − − − MZ2-Mel 3.1 + + − − −SK-MEL-13 − − − − − SK-MEL-19 − − − − − SK-MEL-23 − − − − − SK-MEL-29 −− − − − SK-MEL-30 −* −* − −* − SK-MEL-31 − − − − − SK-MEL-33 − − − − −SK-MEL-37 + + − + + SK-MEL-179 − − − − − M24-MET − − − − − *Positive (+)denotes strong expression. Weak positivity was observed inconsistentlyinSK-MEL-30 for SSX 1,2, and 4, likely representing low levelexpression.

EXAMPLE 7

Additional experiments were carried out to analyze expression of themembers of the SSX family in various tumors. To do this, total cellularRNA was extracted from frozen tissue specimens using guanidiumisothiocyanate for denaturation followed by acidic phenol extraction andisopropanol precipitation, as described by Chomczynski, et al, Ann.Biochem 162: 156-159 (1987), incorporated by reference. Samples of totalRNA (4 ug) were primed with oligoDT(18) primers, and reversetranscribed, following standard methodologies. The integrity of the cDNAthus obtained was tested via amplifying B-acin transcripts in a 25cycle, standard PCR, as described by Tureci, et al, Canc. Res. 56:4766-4772 (1996).

In order to carry out PCR analyses, the primers listed as SEQ ID NOS:5-14, supra were used, as well as SEQ ID NOS: 17 and 18, i.e.:

ACAGCATTAC CAAGGACAGC AGCCACC GCCAACAGCA AGATGCATAC CAGGGACThese two sequences were each used with both SEQ ID NOS: 6 and 8 inorder to detect the SYT/SSX fusion transcript reported for synovialsarcoma by Clark et al, supra, and Crew, et al, supra. The amplificationwas carried out by amplifying 1 μl of first strand cDNA with 10 pMol ofeach dNTP, and 1.67 mN MgCl₂ in a 30 μl reaction. Following 12 minutesat 94° C. to activate the enzyme, 35 cycles of PCR were performed. Eachcycle consisted of 1 minute for annealing (56° C. for SEQ ID NOS: 7 & 8;67° C. for SEQ ID NOS: 9 & 10; 65° C. for SEQ ID NOS: 11& 12; 60° C. forSEQ ID NOS: 13 & 14; 66° C. for SEQ ID NOS: 15 & 16; 60° C. for SEQ IDNOS: 17 & 8 and 18 & 10), followed by 2 minutes at 72° C., 1 minute at94° C., and a final elongation step at 72° C. for 8 minutes. A 15 μlaliquot of each reaction was size fractionated on a 2% agarose gel,visualized with ethidium bromide staining, and assessed for expectedsize. The expected sizes were 421 base pairs for SEQ ID NOS: 7 & 8; 435base pairs for SEQ ID NOS: 9 & 10; 381 base pairs for SEQ ID NOS 11 &12; 413 base pairs for SEQ ID NOS: 13 & 14, and 324 base pairs for SEQID NOS: 15 & 16. The conditions chosen were stringent, so as to preventcross anneling of primers to other members of the SSX family. Additionalsteps were also taken to ensure that the RT-PCR products were derivedfrom cDNA, and not contaminating DNA. Each experiment was done intriplicate. A total of 325 tumor specimens were analyzed. The resultsare presented in Tables 4 & 5 which follow.

It is to be noted that while most of the SSX positive tumors expressedonly one member of the SSX family, several tumor types showedcoexpression of two or more genes.

Expression of SSX genes in synovial sarcoma was analyzed, because theliterature reports that all synovial sarcoma cases analyzed have beenshown to carry either the SYT/SSX1 or SYT/SSX2 translocation, atbreakpoints flanked by the primer sets discussed herein, i.e., SEQ IDNO: 17/SEQ ID NO: 8; SEQ ID NO: 17/SEQ ID NO: 10; SEQ ID NO. 17/SEQ IDNO: 8; SEQ ID NO: 18/SEQ ID NO: 10. The PCR work described supra showedthat SYT/SSX1 translocations were found in three of the synovial sarcomasamples tested, while SYT/SSX2 was found in one. The one in which it wasfound was also one in which SYT/SSX1 was found. Expression of SSXappeared to be independent of translocation.

TABLE 4 Expression of SSX genes by human neoplasms at lease one Tumorentity Tissues tested SSX1 SSX2 SSX3 SSX4 SSX5 positive % Lymphoma 11 — 4 — — —  4 36 Breast cancer 67 5  5 — 10 — 16 23 Endometrial cancer 8 1  2 — 1 1  1 13 Colorectal cancer 58  3 7 —  9 1 16 27 Ovarian cancer12 — — —  6 —  6 50 Renal cell cancer 22 — 1 — — —  1 4 Malignantmelanoma 37 10 13 — 10 2 16 43 Glioma 31 —  2 —  3 —  5 16 Lung cancer24  1  4 —  1 1  5 21 Stomach cancer 3 — — —  1 — ‘1 33 Prostatic cancer5 —  2 — — —  2 40 Bladder cancer 9  2  4 —  2 —  5 55 Head-Neck cancer14  3  5 —  4  1  8 57 Synovial sarcoma 4 —  2 —  1  1  3 75 Leukemia 23— — — — — 0 0 Leiomyosarcoma 6 — — — — — 0 0 Thyroid cancer 4 — — — — —0 0 Seminoma 2 — — — — — 0 0 Total 325 25 50 0 48  7 89

TABLE 5 Expression pattern of individual SSX genes in SSX-positive tumorsamples.¹ SSX1 SSX2 SSX4 SSX5 Breast Cancer (67 specimens) 51 specimens− − − −  7 specimens − − + −  4 specimens − + − −  2 specimens + − − − 2 specimens + − + −  1 specimen + + + + Melanoma (37 specimens) 21specimens − − − −  5 specimens + + + −  4 specimens − + − −  2 specimens− + + −  1 specimen + − − −  1 specimen + + − −  1 specimen + − + −  1specimen + − + +  1 specimen + + + + Endomet. Cancer (8 specimens)  7specimens − − − −  1 specimen + + + + Glioma (31 specimens) 25 specimens− − − −  3 specimens − + − −  2 specimens − − + − Lung Cancer (24specimens) 19 specimens − − − −  3 specimens − + − −  1 specimen − − − + 1 specimen + + + − Colorectal Cancer (58 specimens) 42 specimens − − −−  7 specimens − + − −  5 specimens − − + −  3 specimens + − + −  1specimen − − + + Bladder Cancer (9 specimens)  4 specimens − − − −  2specimens − + − −  1 specimen − − + −  1 specimen + + − −  1specimen + + + − Head-Neck Cancer (14 specimens)  6 specimens − − − −  2specimens + − − −  2 specimens − + + −  1 specimen − + − −  1 specimen −− + −  1 specimen + + − −  1 specimen − + + + SSX1 SSX2 SSX4 SSX5SYT/SSX1 SYT/SSX5 Synovial Sarcoma (4 specimens) Sy1 − − + − + − Sy2 − +− + + − Sy3 − − − − − + Sy4 − + − − + −

EXAMPLE 8

This example details further experiments designed to identify additionalpeptides which bind to HLA-A2 molecules, and which stimulate CTLproliferation.

First, peripheral blood mononuclear cells (“PBMCs” hereafter) wereisolated from the blood of healthy HLA-A*0201⁺ donors, using standardFicoll-Hypaque methods. These PBMCs were then treated to separateadherent monocytes from non-adherent peripheral blood lymphocytes(“PBLs”), by incubating the cells for 1-2 hours, at 37° C., on plasticsurfaces. Any non-adherent PBLs were cryopreserved until needed infurther experiments. The adherent cells were stimulated to differentiateinto dendritic cells by incubating them in AIMV medium supplemented with1000 U/ml of IL-4, and 1000 U/ml of GM-CSF. The cells were incubated for5 days.

Seven days after incubation began, samples of the dendritic cells(8×10⁵) were loaded with 50 μg/ml of exogenously added peptide. (Detailsof the peptides are provided infra). Loading continued for 2 hours, at37° C., in a medium which contained 1000 U/ml of TNF-α, and 10,000 U/mlIL-1β. The peptide pulsed dendritic cells were then washed, twice, inexcess, peptide free medium. Autologous PBLs, obtained as described,supra, were thawed, and 4×10⁷ PBLs were then combined with 8×10⁵ peptideleaded dendritic cells, (ratio: 50:1), in a medium which contained 5ng/ml of IL-7 and 20 U/ml of IL-2. The cultures were then incubated at37° C.

Lymphocyte cultures were restimulated at 14, 21, and 28 days, in thesame manner as the experiment carried out after 7 days. Cytotoxicityassays were carried out, at 14, 21, and 28 days, using a europiumrelease assay, as described by Blomberg, et al., J. Immunol. Meth. 114:191-195 (1988), incorporated by reference, or the commercially availableELISPOT assay, which measures IFN-γ release.

The peptides which were tested were all derived from the amino acidsequence of NY-ESO-1 as is described in U.S. Pat. No. 5,804,381, toChen, et al., incorporated by reference, or the amino acid sequences ofSSX-4. The peptides tested were:

RLLEFYLAM and (SEQ ID NO:  ) SLAQDAPPL (SEQ ID NO:  )both of which are derived from NY-ESO-1, and

-   -   STLEKINKT (SEQ ID NO: 21)        derived from SSX-4. The two NY-ESO-1 derived peptides were        tested in ELISPOT assays. The results follow. In summary, three        experiments were carried out. The results are presented in terms        of the number of spots (positives) secured when the HLA-A2        positive cells were pulsed with the peptide minus the number of        spots obtained using non-pulsed cells. As indicated,        measurements were taken at 14, 21 and 28 days.

The following results are for peptide RLLEFYLAM.

Day Measured (Pulsed Cells - Unpulsed Cells) 14 21 28 Expt 1 30 8 * Expt2 22 * 12 Expt 3 6 * 12 * not determined

EXAMPLE 9

In follow up experiments, the T cell cultures described supra weretested on both COS cells which had been transfected with HLA-A*0201encoding cDNA and were pulsed with endogenous peptide, as describedsupra, or COS cells which had been transfected with both HLA-A*0201 andNY-ESO-1 encoding sequences. Again, the ELISPOT assay was used, for bothtypes of COS transfectants. Six different cultures of T cells weretested, in two experiments per culture.

Pulsed with Endogenous Peptide NY-ESO-1 Production Culture 1 Expt 1 6444 Expt 2 44 52 Culture 2 Expt 1 48 45 Expt 2 100 64 Culture 3 Expt 1 2037 Expt 2 16 16 Culture 4 Expt 1 17 40 Expt 2 28 34 Culture 5 Expt 1 3626 Expt 2 4 36 Culture 6 Expt 1 12 62 Expt 2 44 96The fact that the endogenous NY-ESO-1 led to lysis suggests thatNY-ESO-1 is processed to this peptide via HLA-A2 positive cells.

Similar experiments were carried out with the second NY-ESO-1 derivedpeptide, i.e., SLAQDAPPL. These results follow:

Pulsed with Endogenous Peptide NY-ESO-1 Production Culture 1 Expt 1 2816 Expt 2 30 14 Culture 2 Expt 1 31 75 Expt 2 30 70 Culture 3 Expt 1 3244

EXAMPLE 10

In further experiments, the specificity of the CTLs generated in theprior experiment was tested by combining these CTLs with COS cells,transfected with HLA-A*0201 encoding sequences, which were then pulsedwith peptide. First, the peptide RLLEFYLAM was tested, in threeexperiments, and then SLAQDAPPL was tested, in six experiments. Europiumrelease was measured, as described supra, and the percent of targetcells lysed was determined. The results follow:

% LYSIS Peptide Added No Peptide PEPTIDE RLLEFYLAM Expt 1 43 0 Expt 2 80 Expt 3 9 0 PEPTIDE SLAQDAPPL Expt 1 11 0 Expt 2 13 0 Expt 3 13 0 Expt4 21 0 Expt 5 12 0 Expt 6 42 0In additional experiments, the CTLs specific to RLLEFYLAM/HLA-A2complexes also recognized and lysed melanoma cell line SK-Mel-37 whichis known to express both HLA-A2 and NY-ESO-1. This recognition wasinhibited via preincubating the target cells with an HLA-A2 bindingmonoclonal antibody, BB7.2. This confirmed that the CTLs were HLA-A2specific for the complexes of the peptide and HLA-A2.

EXAMPLE 11

An additional peptide derived from SSX-4, i.e., STLEKINKT (SEQ ID NO:21) was also tested, in the same way the NY-ESO-1 derived peptides weretested. First, ELISPOT assays were carried out, using COS cells whichexpressed KLA-A*0201, and which either expressed full length SSX-4, dueto transfection with cDNA encoding the protein, or which were pulsedwith the peptide. Three cultures were tested, in two experiments. Theresults follow:

Pulsed With Endogenous Peptide NY-ESO-1 Production Culture 1 Expt 1 50100 Expt 2 20 138 Culture 2 Expt 1 8 12 Expt 2 6 14 Culture 3 Expt 1 1547 Expt 2 14 54Further, as with the NY-ESO-1 peptides, specificity of the CTLs wasconfirmed, using the same assay as described supra, i.e., combining theCTLs generated against the complexes with COS cells, transfected withHLA-A*0201, and pulsed with peptide. The europium release assaydescribed supra was used. The results follow:

% LYSIS Peptide Added No Peptide Expt 1 22 0 Expt 2 14 0 Expt 3 46 0Expt 4 16 0As with the NY-ESO-1 derived peptides, CTL recognition was inhibited viapreincubation with the monoclonal antibody BB7.2, confirming specificityof the CTL for complexes HLA-A2 and peptides.

EXAMPLE 12

Additional experiments were carried out on peptides derived from SSX-2i.e, KASEKIFYV, and peptides derived from NY-ESO-1, i.e., SLLMWITQCFL,SLLMWITQC, and QLSLLMWIT (SEQ ID NOS: 72, and 130-132. In each case, thesame type of assays as were carried out in examples 8-11 were carriedout. The results were comparable, in that for each peptide, CTL weregenerated which were specific for the respective peptide/HLA-A2 complex.

EXAMPLE 13

The amino acid sequence of the proteins encoded by the SSX genes wereanalyzed for peptide sequences which correspond to HLA binding motifs.This was done using the algorithm taught by Parker et al., J. Immunol.142: 163 (1994), incorporated by reference, augmented by using, as anadditional motif, nonamers where position 2 is Thr or Ala, and position9 is Thr or Ala. In the information which follows, the amino acidsequence, the HLA molecule to which it presumably binds, and thepositions in the relevant SSX molecule are given. The resultingcomplexes should provoke a cytolytic T cell response. This could bedetermined by one skilled in the art following methods taught by, e.g.,van der Bruggen, et al., J. Eur. J. Immunol. 24: 3038-3043 (1994),incorporated by reference, as well as the protocols set forth inExamples 8-11, supra.

SSX-5 A2 KASEKIIYV 41-49 DAFVRRPRV  5-13 QIPQKMQKA 16-24 MTKLGFKAT 58-66MTFGRLQGI  99-107 NTSEKVNKT 146-154 YVYMKRKYEA 48-57 YMKRKYEAMT 50-59EAMTKLGFKA 56-65 MTKLGFKATL 58-67 RLQGIGPKIT 103-112 QLRPSGKLNT 138-147A3 GIFPKITPEK 106-115 KLNTSEKVNK 144-153 A24 KYEAMTKLGF 54-63 B7HPQMTFGRL  96-104 GPQNNGKQL 131-139 B8 RVRERKQL 167-174 B44 YEAMTKLGF55-63 RERKQLVIY 169-177 B52 KQLVIYEEI 172-180 MTFGRLQGIF  99-108 SSX-4A2 KSSEKIVYV 41-49 VMTKLGFKV 57-65 YVYMKLNYEV 48-57 KLNYEVMTKL 52-61FARRPRDDA  7-13 QISEKLRKA 16-24 MTFGSLQRI  99-107 SLQRIFPKI 103-111KIVYVYMKL 45-53 KLRKAFDDI 20-28 KLRKAFDDIA 20-29 YMKLNYEVMT 50-59MTKLGFKVTL 58-67 QLCPPGNPST 138-147 A3 KLNYEVMTK 52-60 A24 NYEVMTKLGF54-63 B7 RPQMTFGSL  96-104 KPAEEENGL 115-123 GPQNDGKQL 131-139 CPPGNPSTL140-148 B8 RLRERKQL 167-174 B35 RPRDDAQI 10-17 KPAEEENGL 115-123 B44YEVMTKLGF 55-63 RERKQLVVY 169-177 B52 KQLVVYEEI 172-180 MTFGSLQRIF 99-108 SSX-2 A2 KIQKAFDDI 20-28 KASEKIFYV 41-49 AMTKLGFKA 57-65RLQGISPKI 103-111 RLRERKQLV 167-175 DAFARRPTV  5-13 FARRPTVGA  7-15QIPEKIQKA 16-24 MTFGRLQGI  99-107 ELCPPGKPT 138-146 YVYMKRKYEA 48-57EAMTKLGFKA 56-65 MTKLGFKATL 58-67 RAEDFQGNDL 75-84 ELCPPGKPTT 138-147 A3TLPPFMCNK 66-74 KIFYVYMKRK 45-54 A24 KYEAMTKLGF 54-63 B7 RPQMTFGRL 96-104 GPQNDGKEL 131-139 B8 RLRERKQL 167-174 B35 FSKEEWEKM 32-40 B44YEAMTKLGF 55-63 RERKQLVIY 169-177 B52 LQGISPKIM 104-112 KQLVIYEEI172-180 SSX-1 A2 AMTKLGEKV 57-65 AMTKLGFKV 56-65 FAKRPRDDA  7-15KASEKRSKA 16-24 YVYMKRNYKA 48-57 KAMTKLGFKV 56-65 MTKLGFKVT 58-66MTKLGFKVTL 58-67 RIQVEHPQMT  91-100 MTFGRLHRI  99-107 A3 TLPPFMCNK 66-74A24 NYKAMTKLGF 54-63 B7 HPQMTFGRL  96-104 GPQNDGKOL 131-139 B8 RLRERKQL167-174 B44 RERKQLVIY 169-177 B52 KQLVIYEEI 172-180 MTFGRLHRII  99-108NY-ESO-1 A2 SISSCLQQL 148-156 GTGGSTGDA  7-15 RASGPGGGA 52-60 GARGPESRL79-87 ATPMEAELA  97-105 FTVSGNILT 126-134 LTAADHRQL 137-145 QLSLLMWIT155-163 LMWITQCFL 159-167 FATPMEAEL  96-104 TVSGNILTI 127-135 ATGGRGPRGA39-48 GAPRGPHGGA 59-68 LARRSLAQDA 104-113 ITQCFLPVFL 162-171

The foregoing examples describe the isolation and cloning of nucleicacid molecules for the SSX4, splice variant of SSX4, and SSX5 genes aswell as methods for determining expression of the various SSX genes as apossible indication of cancer. As was indicated, supra, these genes areexpressed in tumor cells, thereby enabling the skilled artisan toutilize these for, e.g., assaying for cancer. The determination ofexpression can be carried out via, e.g., determination of transcripts ofan SSX gene or genes, via nucleic acid hybridization, such as viapolymerase chain reaction. In a preferred embodiment, one determinespresence of a transcript of an SSX gene by contacting a sample with anucleic acid molecule which specifically hybridizes to the transcript.

The hybridization of the nucleic acid molecule to a target is indicativeof expression of an SSX gene, and of the possibility of cancer.Preferably, this is done with two primer molecules, as in a polymerasechain reaction. Determination of expression of more than one SSX gene inthe context by these assays also a part of the invention. For theconvenience of the artisan, the nucleotide sequences of SSX 1 and SSX2,which are known, are presented herein as SEQ ID NOS: 1 & 2.

Alternate assays are also a part of the invention. Members of the CTfamily are known to provoke antibodies in the individual who expresses aCT family member. Hence, one can carry out the assays described hereinvia, e.g., determining antibodies in a sample taken from a subject inquestion. Most preferably, the sample being analyzed is serum. Suchassays can be carried out in any of the standard ways one determinesantibodies, such as by contacting the sample with an amount of proteinor proteins, and any additional reagents necessary to determine whetheror not the antibody binds. One approach involves the use of immobilizedprotein, where the protein is immobilized in any of the standard waysknown to the art, followed by contact with the sample and then, e.g.,anti-IgG, anti-Fc antibodies, and so forth. Conversely, presence of anSSX protein can also be determined, using antibodies in the place of theproteins of the above described assays.

The correlation of SSX expression with cancer also suggests varioustherapeutic methods and compositions useful in treating conditionsassociated with abnormal SSX expression. “Abnormal SSX expression” inthis context may mean expression per se, or levels which differ fromthose in a normal individual, i.e., they may be lower or higher.

The invention envisions therapeutic approaches such as the use ofantisense molecules to inhibit or block expression. This antisensemolecules are oligonucleotides which hybridize to the nucleic acidmolecules and inhibit their expression. Preferably these are 17-50nucleotides in length. These antisense oligonucleotides are preferablyadministered in combination with a suitable carrier, such as a cationicliposome.

Other therapeutic approaches include the administration of SSX proteinsper se, one or more antigenic peptides derived therefrom, as well asso-called polytopic vaccines. These include a plurality of antigenicpeptides, untied together, preferably by linker sequences. The resultingpeptides may bind to either MHC-Class I or Class II molecules. Theseproteins, peptides, or polytopic vaccines may be administered incombination with an appropriate adjuvant. They may also be administeredin the form of genetic constructs which are designed to permitexpression of the protein, the peptide, the polytopic structures, etc.Peptides and polytopic structures can be expressed by so-called“minigenes” i.e., DNA molecules designed to express portions of theentire SSX molecule, or the various portions of the molecules, linkedtogether as described supra. One can formulate the therapeuticcompositions and approaches described herein such that one, or more thanone SSX protein, is used as the source of the compositions. In otherwords, if a whole protein approach is used, one SSX molecule may beused, or two or more may be combined in one formulation. For peptides,these can all be taken from one SSX molecule, or be combinations ofpeptides taken from more than one. The polytopic structures describedherein can also be made up of components of one, or more than one, SSXmolecule.

The amount of agent administered and the manner in which it isadministered will, vary, based on the condition being treated and theindividual. Standard forms of administration, such as intravenous,intradermal, subcutaneous, oral, rectal and transdermal administrationcan be used. With respect to formulations, the proteins and or peptidesmay be combined with adjuvant and/or carriers such as a saponin, GM-CSF,one or more interleukin, an emulsifying oil such as vitamin E, one ormore heat shock protein, etc.

When the nucleic acid approach is utilized, various vectors, such asVaccinia or adenovirus based vectors can be used. Any vector useful ineukaryotic transfection, such as in transfection of human cells, can beused. These vectors can be used to produce, e.g., cells such asdendritic cells which present relevant peptide/MHC complexes on theirsurface. The cells can then be rendered non-proliferative prior to theiradministration, using standard methodologies.

Also a part of the invention are peptides which consist of amino acidsequences corresponding to portions of SSX molecules, or the NY-ESO-1molecule, such as those peptide sequences described supra. As has beenshown, such peptides bind to MHC molecules, such as HLA-A2 molecules,and provoke proliferation of cytolytic T cells against the formedcomplexes. As it has been shown that cells which express the full lengthmolecules (NY-ESO-1, or SSX molecules) are in fact recognized by CTLswhich were generated following pulsing of cells with relevant peptides.This result indicates that both the peptides and CTLs should be usefultherapeutic agents. Hence, an additional aspect of the invention is theadministration of one or more peptides, derived from NY-ESO-1 or an SSXmolecule as described, alone or in combination, such as in antigen“cocktails.” Such cocktails can include a mixture of peptides, whichhave been formulated following typing of a particular patient's HLAtype. Similarly, CTLs, developed in vitro, can be administered to thepatient, in view of the recognition that the peptides are presentedfollowing endogenous expression of the full length molecule.

It is to be pointed out that when an MHC molecule is mentioned, such asHLA-A2, this is meant to include all allelic forms of that molecule.There are various types of HLA-A2 molecules which are known, and whilethese differ in a few amino acids, the degree of disparity is generallyless than 10 amino acids over the full length of the molecule, and thedifferences are not expected to impact the ability of the form of themolecule to bind to peptides. Hence, a peptide which binds to anHLA-A*0201 molecule may by presumed to also bind to HLA-A*0202,HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0209, and soforth.

Other aspects of the invention will be clear to the skilled artisan andneed not be reiterated herein.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

1. An isolated nucleic acid molecule consisting of a nucleotide sequencewhich encodes an isolated peptide consisting of the amino acid sequenceset forth at SEQ ID NO:44.
 2. An expression vector comprising theisolated nucleic acid molecule of claim 1 operably linked to a promoter.3. An isolated recombinant cell comprising the isolated nucleic acidmolecule of claim
 1. 4. The recombinant cell of claim 3, furthercomprising a nucleic acid molecule which encodes an HLA molecule.